Transgenic plant product development by conventional transformation and breeding efforts is a slow and unpredictable process. Gene targeting systems can overcome problems with expression variability, unpredictable of impacts of random gene insertion on agronomic performance, and the large number of experiments that need to be conducted. Such systems can also provide approaches to manipulating endogenous genes. Of course, targeting systems require the ability to focus the recombination process to favor recovery of desired targeting events.
The natural cellular DNA repair and recombination machinery consists of a complex array of protein components interacting in a highly controlled manner to ensure that the fidelity of the genome is conserved throughout the many internal events or external stimuli experienced during each cell cycle. The ability to manipulate this machinery requires an understanding of how specific proteins are involved in the process, and how the genes that encode those proteins are regulated. Since the primary approaches to gene targeting involve recombinases, whether operating in their natural in vivo environment (as during normal recombination) or as part of schemes that involve pretreatment of substrates so as to associate DNA with a recombinase and increase efficiency of targeting (e.g., double D-loop), there is a continuing need to isolate and characterize the genes for these molecules. Because many different protein components may be involved in gene targeting, the availability of host-specific genes and proteins could avoid possible problems of incompatibility associated with molecular interactions due to heterologous components.
The RAD6 gene of the baker's yeast Saccharomyces cerevisiae encodes a protein (designated Rad6) which is involved in DNA repair. The Rad6 protein exhibits ubiquitin conjugating activity. Hence, it is also known as Ubiquitin Conjugating Enzyme (UBC) or E2. The RAD6 gene belongs to a large family which includes many closely related members. RAD6 or E2 has been cloned from many eukaryotic organisms such as worm (C. elegans), baker's yeast (S. cerevisiae), budding yeast (S. pombe), fruitfly (D. melanogaster), and humans. Members of the RAD6 family have also reportedly been cloned from wheat, Arabidopsis, tomato, rice, peas and maize. (Stary et al., Curr. Genet. 32:309-314, 1997; Gray et al., Plant Physiol., 103:1471-1472, 1993; Feussner et al., FEBS Lett. 409:211-215, 1997; Chen and Wang, Plant Mol. Biol., 29: 787-795, 1995; Woo et al., Gene, 148:369-370, 1994; Sullivan and Vierstra, PNAS (USA) 86: 9861-9865, 1989). The RAD6 gene has been highly conserved in eukaryotic evolution.
RAD6 yeast mutants show very diverse phenotypes indicating the involvement of this gene in multiple cellular functions. Montelone et al. showed that a point mutant, rad6-1, is proficient in spontaneous and UV induced unequal sister chromatid recombination, but deficient in UV induced mutagenesis, sporulation and meiotic recombination (Montelone B A et al., Mol. Gen. Genet, 184(3):410-415, 1981). These workers also found that another point mutant, rad6-3, shared some phenotypes with the rad6-1 mutant, except that it was not sporulation defective, indicating a specificity of functions. The sporulation defect in rad6-1 cells appears to be downstream of the premeiotic DNA synthesis but prior to meiotic recombination (Montelone B A et al., Mol. Gen. Genet, 184(3):410-415, 1981). Yeast RAD6 deletion mutants also show a sporulation defect (Game J C et al., Genetics, 94: 51-68, 1980). A homologue of RAD6 (termed RPH6.sup.+) has been identified in the budding yeast Schizosaccharomyces pombe (Reynolds P et al., EMBO J, 9:1423-30, 1990). The two genes from these distantly related yeast share a high degree of structural and functional homology. Thus, like rad6, null mutations of the rhp6+ gene confer a defect in DNA repair, UV mutagenesis and sporulation, and the RAD6 and rhp6+ genes can functionally substitute for one another.
Roest et al. (Roest et al., Cell, 86: 799-810, 1996) generated transgenic mice that were null mutants for RAD6 gene. These mice did not have any detectable levels of RAD6 message or protein. Interestingly and quite unexpectedly, it was observed that the male RAD6 null mice were infertile because of impaired spermatogenesis. More surprisingly, heterozygous males or homozygous females did not show any abnormality with respect to fertility. These findings further substantiated the role of RAD6 genes in inhibiting yeast sporulation.
To date, work with recombinase enzymes in plants, however, has been very limited. Accordingly, there is an ongoing need for the identification and characterization of the functional activities of RAD6 to provide improved and expanded methods of gene targeting in plant systems or for the creation of male sterile plants, particularly in agriculturally important crop species such as maize. The present invention provides this and other advantages.